This invention presents a method of alleviating pain, such as central pain, pain arising from cancer and phantom limb pain, by systemic administration of sodium channel blocking compounds, including tetrodotoxin and saxitoxin.
Pain is a sensation that hurts. It may cause discomfort or distress or agony. It may be steady or throbbing. It may be stabbing, aching, or pinching. However pain is felt, only the person experiencing pain can describe it or define it. Because pain is so individual, pain cannot be truly evaluated by any third person.
The World Health Organization (WHO) recognizes a xe2x80x9cThree-step Analgesic Ladderxe2x80x9d for pharmacologic management of pain. The ladder begins with relatively low doses of low-potency analgesics and progresses to higher doses of more potent compounds. The three steps involve use of:
Nonopioid analgesics with or without coanalgesics, such as Nonsteroid Anti-inflammatory Drugs (NSAIDs);
Lower-potency opioids with or without coanalgesics as pain persists or increases to moderate levels;
High-potency opioids with or without nonopioid coanalgesics as pain persists or increases to severe levels.
Use of opioid analgesics, even for treatment of severe pain, is controversial in the medical community, due to the possibility of addiction. See, e.g. S. E. Weitz et al., New Jersey Medicine, Vol. 97: 63-67 (2000).
Tetrodotoxin is a nonprotein neurotoxin that is found in multiple diverse animal species, including puffer fish, goby fish, newt, frogs and the blue-ringed octopus.
Tetrodotoxin can be obtained from the ovaries and eggs of several species of pufferfish of the suborder Gymnodontes. Tetrodotoxin is the agent for poisoning occurring from the consumption of ill-prepared fugu fish in sushi bars. Tetrodotoxin can also be obtained from California newts of the genus taricha.
One biological activity of tetrodotoxin is binding of the alpha subunit of neuronal sodium channels. Tetrodotoxin has a chemical formula of C 11H17N3O8, and has a molecular weight of 319.28. The Merck Index, 10th Ed. (1983), states tetrodotoxin is the generic name for the compound octahydro-12-(hydroxymethyl)-2-imino-5,9:7,10a-dimethano-10aH-(1,3)dioxocino(6,5-d)-pyrimidine-4,7,10,11,12-pentol, which has the following structure: 
The Merck Index, 10th Ed. (1983), states tetrodotoxin is also referred to as maculotoxin, spheroidine, tarichatoxin, tetrodontoxin, fugu poison and TTX.
According to U.S. Pat. No. 6,030,974, xe2x80x9ctetrodotoxinxe2x80x9d or xe2x80x9cTTXxe2x80x9d refers to the amino perhydroquinazoline compounds having the molecular formula C11H17N3O8 and to derivatives thereof, including but not limited to anhydrotetrodotoxin, tetrodaminotoxin, methoxytetrodotoxin, ethoxytetrodotoxin, deoxytetrodotoxin and tetrodonic acid (Kao). Examples of TTX analogs include novel TTX analogs isolated from various organisms, as well as those that are partially or totally chemically synthesized. See e.g., Yotsu, M. et al. Agric. Biol. Chem., 53(3):893-895 (1989). Such analogs bind to the same site on the alpha subunit of sodium channels as does TTX.
Adams, et al., U.S. Pat. Nos. 4.022,899 and 4,029,793 pertain to a local anesthetic composition comprising a mixture in a pharmaceutically acceptable carrier of a particular toxin, namely tetrodotoxin or desoxytetrodotoxin, and another compound, generally a conventional local anesthetic compound or a similar compound having nerve-blocking properties. The conventional local anesthetic can be an aminoacylanilide such as lidocaine, an aminoalkylbenzoate such as procaine, cocaine, an amino carbamate such as diperodon, a N-phenylamidine such as phenacine, a N-aminoalkyl amide such as dibucaine, an aminoketone such as falicain, or an aminoether such as pramoxine.
According to U.S. Pat. No. 6,030,974, xe2x80x9csaxitoxinxe2x80x9d or xe2x80x9cSTXxe2x80x9d refers to a compound comprising a tetrahydropurine moiety composed of two guanidine units fused together in a stable azaketal linkage, having a molecular formula C10H17N7O4, (mol. wt. 299.30) and to derivatives thereof, including but not limited to hydroxysaxitoxins and neosaxitoxin. See Bower et al., Nonprotein Neurotoxins, Clin. Toxicol. 18(7):813-863 (1981).
Tetrodotoxin and its significance in the study of excitation phenomena has been reviewed by C. Y. Kao, Pharmacological Reviews, Vol. 18, No. 2, 997-1049 (1966). Kao notes that one of the most prominent actions of tetrodotoxin in the whole animal is a rapidly progressive and marked weakening of all voluntary muscles, including the respiratory muscles (Kao at 1016). However, Kao notes that specific action of tetrodotoxin on the central nervous system is debatable (Kao at 1022, line 3).
Pan et al., U.S. Pat. No. 5,846,975, discloses the use of amino hydrogenated quinazoline compounds, such as tetrodotoxin, for treating drug dependence in humans. Tetrodotoxin was shown to be effective against withdrawal symptoms from opium, heroin, morphine, cocaine, amphetamine, dolandin, dihydroetorphine and methadone. Amounts effective for relieving withdrawal symptoms are described in this patent.
Tetrodotoxin can be used as a local anesthetic and is ten thousand times more powerful than commonly used local non-narcotics, as is discussed by C. Y. Kao and F. A. Fuhrman, J. Pharmacol., 140, 31-40 (1963). Tetrodotoxin preparations in combination with other widely used anesthetics have been noted in U.S. Pat. Nos. 4,022,899 and 4,029,793.
U.S. Pat. No. 6,030,974 describes a method of producing local anesthesia in a mammal experiencing pain in an epithelial tissue region. The method includes topically administering to the region, in a suitable pharmaceutical vehicle, an effective dose of a long-acting sodium channel blocking compound. The sodium channel blocking compound of U.S. Pat. No. 6,030,974 can be a formulation of tetrodotoxin or saxitoxin at a concentration of between 0.001-10 mM.
Zapata et al., Pain 72:41-49 (1997) discusses the utilization of tetrodotoxin for the inhibition of neuropathic ectopic activity in neuromas, dorsal root ganglia and dorsal horn neurons. The neuronal activity arises from neuroma caused by mechanical, chemical or ischemic injury. The effect of intravenously administered TTX on the neuronal induction by sciatic nerves in male rats was researched. However, the dosages and effects studied by Zapata et al. were applied to animals under anesthesia and artificial ventilation, thus these doses are above the maximal tolerated dose and the administration was under conditions that are not applicable to the presently intended clinical use of tetrodotoxin.
Although there has been extensive research into the effectiveness of TTX and its derivatives as a sodium channel blocker and local anesthetic, systemic administration of pure TTX as an analgesic has never been disclosed. The potential for TTX to alleviate pain arising from the activity in the central nervous system, rather from the stimulation of peripheral nerves does not seem to have been described.
The alleviation of chronic severe pain, such as that arising from cancer and xe2x80x9cphantom limb painxe2x80x9d is an important topic in modern medicine. Cancer is highly pervasive in the human population.
A person suffering from cancer frequently experiences severe pain. This pain can also be known as central pain or chronic pain. However, a patient need not be suffering from cancer to experience central pain or chronic pain. A related type of pain is phantom limb pain. These types of pain have been treated by opiates such as morphine. A drawback of the opiate analgesia is the addictive nature of the opiates.
Acute local pain can arise, for example, from toothaches, eye irritation, inflammation in a nervous tissue region, canker sores, genital ulcers, and pain in epithelial tissues caused by burns, surgery or soreness.
Perception of pain can also be divided into three areas; acute nociceptive processing, facilitated pain arising from persistent afferent input (as after tissue injury) and neuropathic pain that arises from altered processing after nerve injury.
Some sodium channel blocking compounds, e.g. lidocaine and mexiletine, typically used as local anesthetics, have also been administered systemically. These compounds seem to be marginally effective in blocking acute nociceptive processing, and there is some effect observable upon spinal processing and substance P release, indicating effects on facilitated pain. However, the effective doses are above the maximum tolerated dose and thus side effects have precluded use of these compounds as systemic analgesics. Furthermore, sodium channel blockers have previously been found entirely ineffective in managing neuropathic pain. See, M. S. Wallace, xe2x80x9cCalcium and Sodium channel blocking compounds for the Treatment of Painxe2x80x9d, Clin. J. Pain, Vol. 16: S80-S85 (2000).
Several sodium channel blockers such as lidocaine and carbamazepine have been used in the treatment of neuropathic pain and trigeminal neuralgia. These substances may block sodium channels to abolish abnormal peripheral nerves activity at concentrations which do not block nerve conduction. Since it may cause severe damage to the function of liver, however, carbamazepine should be restricted from being used on women in the early stage of pregnancy and during breasting period, and should be used with caution on older people and those who have glaucoma or severe angiocardiopathy. On the other hand, lidocaine has such an excitation effect on the central nerve system that it can cause tremor, shivering and clonic spasm. Therefore, these two drugs are considered inappropriate to promote as new analgesics for systemic use. This has stimulated interest in developing other sodium channel blocking drugs.
In 1998, Rabert et al demonstrated that the existence of more than one type of sodium channels in rat dorsal root ganglion (DRG) sensory neurons. These sodium channels have been distinguished on the basis of a differential sensitivity to TTX: a TTX-sensitive sodium channel (TTX-S) is blocked by TTX with IC50 of 1-20 nM. A TTX-resistant sodium channel (TTX-R) is blocked by TTX with an IC50 of xcx9c100 xcexcM. The rBIIA, rBII1, rSKM1, rPN1 and rPN4 sodium channels are all TTX-S, whereas rPN3/SNS sodium channels are TTX-R. There are also two types of sodium channels in human DRG sensory neurons: hPN1 is a TTX-S channel and hPN3 is a TTX-R channel blocked by TTX with an IC50=80 xcexcM. Rabbert also showed that sodium channels in mammalian DRG sensory neurons express at least two sodium currents: a TTX-sensitive current (TTX-SINa) with rapid inactivation kinetics and a TTX-resistant current (TTX-RINa) with slower inactivation kinetics. The biological role of the two sodium currents has not been delineated whereas numerous studies indicated that the properties of the TTX-RINa currents in dorsal root ganglia appear well suited to contribute to the sustained neuronal firing characteristic of most neuropathic pain conditions.
Nociceptors are primary afferent neurons that respond to noxious or potentially tissue-damaging stimuli and are unique among sensory neurons because they can be sensitized. The decrease in the threshold and increase in the response to a constant stimulus that are characteristic of nociceptor sensitization are thought to underlie the hyperalgesia or tenderness associated with tissue injury. Agents released at the site of tissue injury sensitize nociceptors by initiating a cascade of event that likely results in a change in ionic conductance of the nociceptor peripheral terminal. Small-diameter sensory neurons in the DRG are known to express a TTX-R channel activity. A variety of inflammatory insults and direct damage to sensory neuron fibers produce a decrease in the thresholds of activation of sensory neurons, while prolonged activation of sensory neurons can lead to central sensitization to noxious input within the spinal cord. When sensory neurons were highly excited, activity of sodium channels and voltage-gated sodium current were increased significantly. Recent numerous studies suggest that increase of TTX-RINa may play a significant role in the hyperexcitability of sensory neurons. Increased TTX-RINa may contribute to diverse acute and chronic pain such as neuropathic pain and neuroma pain which were induced by inflammation and nerve damage. Patch-clamp electrophysiological techniques have been used to study the effects of hyperalgesic agents that modulate TTX-RINa at primary culture DRG neurons. Evidence suggests that prostaglandin E2 (PGE2), adenosine and serotonin increase the magnitude of TTX-RINa, shift its conductance-voltage relationship in a hyperpolarized direction, and increase its rate of activation and inactivation. In contrast, thromboxane B2, a cyclooxygenase product which does not produce hyperalgesia, does not affect TTX-RINa. These results suggest that an increase in TTX-RINa underlies the increase in nociceptor neuronal sensitization induced by hyperalgesic agents. Intratheacal administration of antisense and sense oligodeoxynucleotides (ODNS), which were directed against a unique sequence of the rPN3 or SNS were used to examine the role of these channels in PGE2-induced hyperalgesia. Only antisense ODNs led to a decrease in PGE2-induced hyperalgesia. PGE2-induced hyperalgesia was partially recovered 4 days after the last antisense ODN injection, and was fully recovered within 7 days. Antisense ODNs selectively and significantly reduced TTX-RINa current density in cultured sensory neurons. These finding support the hypothesis that modulation of TTX-RINa contributes to inflammatory hyperalgesia.
Novakovis et al by their immunohistochemical studies, showed that sodium channels, especially PN3 channels, accumulated at the site of injury. The subcellular distribution of PN3 channels also changed after neuropathic injury, and nerve conduction was significantly altered. Sodium channel anterograde axonal transport is completely blocked in neuropathic pain and neuroma pain models, and is significantly reduced in the chronic constriction injury model of neuropathic pain (CCI). Because sodium channels, presumably including TTX-R channels, are constantly being transported to peripheral terminals, alterations in axonal transport ultimately result in channel accumulation at the injury site. Nerve degeneration and subsequent regeneration of many new axonal sprouts could be observed at the injury site in the CCI and neuroma models. Many of these new sprouts appear to be immunopositive for PN3. The overaccumulation of sodium channels occurs in regeneration fibers. Sensitization of CNS is an important characteristic of neuropathic pain. Establishment and maintainance of CNS sensitization relies on sense information conducted by nociceptor nerve fibers. In the pain state, because TTX-R channels are involved in coding information of pain sense, TTX-R channels are thought to play an important role in central perception of pain input.
In summary, modulation of TTX-R sodium channels is thought to play a role in the sensitization of nociceptors in the persistent pain state. The tissue distribution of TTX-R channels is restricted to a subpopulation of sensory neurons with properties of nociceptors. It is possible that designing a pharmacotherapeutic agent that selectively blocks TTX-R channels will be effective for pain relief. hPN3 may prove to be a valuable target for a therapeutic agent for treatment of acute and chronic pain.
TTX blocking of TTX-R channels may contribute to antinociceptive action of TTX in animals. In animal models of pain, neuromas, neuropathic pain or persistent dysesthesis initiated by artificial damage to peripheral sensory nerves produces ectopic discharges originating at both injury site and related dorsal root ganglia, and consequently hyperexcitability in associated dorsal horn (DH) neurons of spinal cord. TTX inhibits neuropathic ectopic activity in neuromas, DRG, and DH neurons in a dose-dependent pattern. However, at present the relative contribution of TTX-S and TTX-R channels to the generation of ectopic discharges in neuromas, DRG, and DH neurons is not clear.
TTX produces antinociceptive action at dose levels that do not significantly change behavior of animals. However, TTX at these dose levels does not modulate distribution and function of sodium channels, nor does TTX fully block nerve conduction in various types of pain conditions. These results suggest that TTX may unexpectedly act on TTX-R sodium channels to produce an antinociceptive action.
Pain may be acute or chronic. Acute pain can be severe, but lasts a relatively short time. It is usually a signal that body tissue is being injured in some way, and the pain generally disappears when the injury heals. Chronic pain may range from mild to severe, and it is present to some degree for long periods of time. Chronic pain often arises without any detectable injury.
TTX is also effective for alleviating acute pain induced by mechanical and chemical stimulation, and inflammation.
Tetrodotoxin (TTX) has been shown to be effective on pains produced by liver cancer, rectal cancer, leiomyosarcoma, bone cancer, stomach cancer, lymphatic cancer, esophageal cancer and other major cancer types. TTX is also effective on central pain, chronic pain and phantom limb pain.
Tetrodotoxin is effective on all severe chronic pains. Tetrodotoxin is capable of generating analgesia in a mammal experiencing acute or chronic pain. The method of the present invention includes systemically (generally, to the whole body) administering, in a suitable pharmaceutical vehicle, an effective dose of a long-acting sodium channel blocking compound, i.e. tetrodotoxin.
TTX is administered in a dosage range of 0.1-1 xcexcg/kg. TTX is administered in a schedule of up to 4 doses per day over a time period of 3 days. Frequently the effectiveness of the dose lasts for up to 20 days.
The purity of TTX is usually 96% or greater.
Saxitoxin (STX) is a highly selective and highly active sodium channel blocking compound. According to U.S. Pat. No. 6,030,974, both TTX and STX specifically bind to a site on an extracellular region of a sodium channel alpha subunit. The site is in either an SS1 region or an SS2 region (Evans, Tetrodotoxin, Saxitoxin, and Related Substances: Their Applications in Neurobiology, International Review of Neurobiology, Vol. 15, pp. 83-166, 1972, Academic Press).
The LD50 of saxitoxin for mice by intraperitoneal injection is 10 xcexcg/kg (Schantz, E. J., McFarren, E. F., Schaeffer, M. L. and Lewis, K. H.: Purified shellfish poison for bioassay standardization. J. Assoc. Official Agricul. Chemist. 41: 160-168, 1958.); in rats, the intraperioneal LD50 is 10.5 microgram/kg (Watts, J. S., DaCosta, F. and Reilly, J.: Some factors influencing the action of paralytic shellfish poison in rats. Fed. Proc. 24: 392, 1965); the estimated human lethal dose (oral) of between 300 xcexcg to 1.0 mg (Bower et al., Clin. Toxicol., 18(7):813-863, 1981).
In view of the similar mode of action and toxicity of TTX and STX, dosages of the two toxins for analgesia are also similar.
Pain can originate for many reasons. A familiar cause is trauma, such as a sprain or muscle injury or broken bone, or from surgery. Pain due to inflammation, such as a toothache, is also familiar to many. Headache is a common experience and arises often for unknown reasons.
Cancer patients may have pain for a variety of reasons. It may be due to the effects of the cancer itself, or it could result from treatment methods. For example, after surgery a person feels pain as a result of the operation itself. Not all people with cancer have pain, and those who do are not in pain all the time.
Cancer pain may depend on the type of cancer, the stage (extent) of the disease, and the patient""s pain threshold (tolerance for pain). Cancer pain that lasts a few days or longer may result from:
The tumor causing pressure on organs, nerves, or bone.
Poor blood circulation because the cancer has blocked blood vessels.
Blockage of an organ or tube in the body.
Metastasisxe2x80x94cancer cells that have spread to other sites in the body.
Infection or inflammation.
Side effects from chemotherapy, radiation therapy, or surgery.
Stiffness from inactivity.
Psychological responses to illness such as tension, depression, or anxiety.
The difference between acute and chronic pain is discussed by Joseph T. Dipiro, xe2x80x9cPharmacotherapy: A Pathophysiologic Approachxe2x80x9d, Third Edition, Appleton and Lange (1997) p. 1263. Dipiro explains that acute pain may be a useful physiologic process warning individuals of disease states and potentially harmful situations. Unfortunately, severe, unremitting, undertreated pain, when it outlives its biologic usefulness, can produce many deleterious effects such as psychological problems. When pain is not effectively treated, the stress and concurrent reflex reactions often cause hypoxia, hypercapnia, hypertension, excessive cardiac activity, and permanent emotional difficulties. The problems associated with these reactions range from prolonged recovery time to death.
Under normal conditions, acute pain quickly subsides as the healing process decreases the pain-producing stimuli. However, in some instances pain may persist for months to years, leading to a chronic pain state with features quite different from those of acute pain. Typically, chronic pain is divided into four subtypes: pain that persists beyond the normal healing time for an acute injury, pain related to a chronic disease, pain without identifiable organic cause, and pain that involves both the chronic and acute pain associated with cancer. Patients in chronic pain often develop severe psychological problems caused by fear and memory of past pain. In additional, chronic pain patients may develop dependence and tolerance to analgesics, have trouble sleeping, and more readily react to environmental changes that can intensify pain and the pain response. Distinguishing between chronic and acute pain states is very important because of differing management techniques.
Acute and chronic pain can also be classified by duration. Acute pain lasts or is anticipated to last less than 1 month, e.g., postoperative pain. Chronic pain is usually defined as pain persisting more than 1 month, e.g., cancer pain and phantom limb pain.
The National Institute of Neurological Disorders and Stroke, National Institutes of Health (http://healthlink/mcw.edu/article/921391401.html; Jun. 29, 2000) describes central pain syndrome as a neurological condition caused by damage specifically to the central nervous system (CNS)xe2x80x94brain, brainstem, or spinal cord. The pain is steady and is usually described as a burning, aching, or cutting sensation. Occasionally there may be brief, intolerable bursts of sharp pain.
Central pain is characterized by a mixture of pain sensations, the most prominent being constant burning. Mingled with the burning are sensations of cold, xe2x80x9cpins and needlesxe2x80x9d tingling, and nerve proximity (like that of a dental probe on an exposed nerve). The steady burning sensation is increased significantly by any light touch. Patients are somewhat numb in the areas affected by this burning pain. The burning and loss of touch appreciation are usually most severe on the distant parts of the body, such as the feet or hands. Pain may be moderate to severe in intensity and is often exacerbated by movement and temperature changes, usually cold temperatures.
Central pain syndrome may develop months or even years after injury or damage to the CNS. The disorder occurs in patients who have, or have had, strokes, multiple sclerosis, limb amputations, or brain or spinal cord injuries.
Generally pain medications provide little or no relief for those affected by central pain syndrome. Patients should be sedated and the nervous system should be kept quiet and as free from stress as possible. Central pain syndrome is not a fatal disorder. But for the majority of patients, the syndrome causes intractable pain.
The best way to manage pain is to treat its cause. For example, whenever possible, the cause of pain from cancer is treated by removing the tumor or decreasing its size. To do this, the doctor may recommend surgery, radiation therapy, or chemotherapy. When none of these procedures can be done, or when the cause of the pain is not known, pain-relief methods are used.
In the past, analgesics were differentiated as peripheral (e.g., aspirin, acetaminophen) and central acting (opioids) analgesics. Due to current better understanding of pain relief and analgesics, it is now more accepted to distinguish between non-opioid and opioid analgesics.
Non-opioid analgesics are often effective for mild to moderate pain and in treating pain arising from rheumatoid arthritis. Typical non-opioid analgesics are aspirin, acetaminophen and other nonsteroid anti-inflammatory drugs (NSAIDs), e.g., ibuprofen, piroxicam, and naproxen.
Opioid (or opiate) is a general term for natural or synthetic substances that bind to specific opioid receptors in the central nervous system producing an agonist action. Opioid analgesics are extremely useful in managing severe acute pain, postoperative pain and chronic pain including cancer pain. Typical opioid analgesics are codeine, morphine, methadone and fentanyl.
Traditional cancer pain relief methods include use of opiates such as codeine, hydromorphone (Dilaudid), levorphanol (Levo-Dromoran), methadone (Dolophine), morphine, oxycodone (in Percodan), and oxymorphone (Numorphan). They may be taken by mouth (orally, or PO), by injection (intramuscularly, or IM), through a vein (intravenously, or IV), or by rectal suppository. There are also other methods of giving pain medicines for more continuous pain relief. Not all narcotics are available in each of these forms.
NSAIDs similar to ibuprofen (in large doses, ibuprofen requires a prescription) are used for treatment of pain from cancer. Included in this group of pain relievers are Motrin, Naprosyn, Nalfon, and Trilisate. They are useful for moderate to severe pain. They may be especially helpful in treating the pain of bone metastasis.
It is believed that tetrodotoxin is not an opioid agonist since it does not bind specific opioid receptors in the CNS (central nervous system) producing an agonist action. Tetrodotoxin is a specific sodium channel blocker. Sodium channel blockers are used as local anesthetics, e.g., lidocaine. It is evident that tetrodotoxin is not an opioid agonist and therefore it could be assigned to the class of the non-opioid analgesics. As a result, tetrodotoxin has the potential to be a very strong non-opioid without a risk for addiction.
The inventors have discovered that tetrodotoxin (TTX), its analogs and derivatives are effective on pains produced by cancers such as liver cancer, rectal cancer, leiomyosarcoma, bone cancer, stomach cancer, lymphatic cancer, esophageal cancer and other major cancer types. Tetrodotoxin and its analogs and derivatives are effective in relieving pain in humans and other mammals resulting from malignant neoplasm (cancers) or other tumors. These cancers can occur in the genital organs (including prostate), digestive system (including stomach, colon), breast, respiratory system (including lung and bronchus), urinary system, lymphoma and skin cancer.
A person who has had an arm or leg removed by surgery may still feel pain or other unpleasant sensations as if they were coming from the absent limb. Doctors are not sure why it occurs, but phantom limb pain is real; it is not imaginary. This also can occur if a patient had a breast removed, resulting in a sensation of pain in the missing breast.
No single pain relief method controls phantom limb pain in all patients all the time. Many methods have been used to treat this type of pain, including pain medicine, physical therapy, and nerve stimulation. Tetrodotoxin administered in accordance with the method of the invention provides relief from the pain associated with phantom limb pain.
Since tetrodotoxin has high physiological activity, strong toxicity and a low safety threshold value, it is necessary to accurately and precisely control the formulation and dosage. Several methods reported in the literature for the determination of tetrodotoxin include biological measurement, UV spectrophotometry, fluorometry, gas chromatography, liquid chromatography, etc. All the techniques have their advantages and limitations. The biological measurement method is very sensitive and considered a feasible technique, however, it also has shortcomings like poor reproducibility, many influential factors, large variance between test animals, and deficiency of objectivity. TLC has relatively large sampling amount (20 xcexcg) and low detection limit. The fluorometry method requires a fluorescence spectrophotometer. UV spectrophotometry cannot separate tetrodotoxin from related impurities, and its accuracy is poor. GC and the electrophoresis method also have their limitations, respectively.
Since it provides high specificity, high sensitivity, and is capable of providing identification and content determinations simultaneously, HPLC is used as the major detection method for content determination. By routine experimentation known to the skilled practitioner, the stationary phase, mobile phases and the detection conditions are optimized to establish a reliable separation and detection method. As a result, tetrodotoxin and the major related substances can be well separated. HPLC methods provide high detection sensitivity, convenience of operation, and sound reproducibility.
Tetrodotoxin useful in the method of the present invention can be obtained from animal tissues, such as puffer fish organs.
A detailed description of a method for production of tetrodotoxin and derivatives thereof is provided in Chinese application no. 00124516.3, filed Sep. 18, 2000.
The typical analogs of TTX possess only xe2x85x9 to {fraction (1/40)} toxicity of TTX, based upon bioassay in mice. It has been observed that the analogs produce joint analgesic action, and do not interact adversely.
The invention pertains to all sodium channel blocking compounds such as tetrodotoxin and saxitoxin. Chiriquitoxin (CTX) can be used. Also effective are analogs of tetrodotoxin such as 4-epi-tetrodotoxin, and anhydro-4-epi-tetrodotoxin.
Tetrodotoxin or TTX refers to the amino perhydroquinazoline compound having the molecular formula C11H17N3O8 and to derivatives thereof, including but not limited to anhydrotetrodotoxin, tetrodaminotoxin, methoxytetrodotoxin, ethoxytetrodotoxin, deoxytetrodotoxin and tetrodonic acid. Examples of TTX analogs include novel TTX analogs isolated from other organisms, as well as those that are partially or totally chemically synthesized. See e.g., Yotsu, M. et al. Agric. Biol. Chem., 53(3):893-895 (1989). Such analogs bind to the same site on the alpha subunit of sodium channels as does TTX.
Saxitoxin or STX refers to a compound comprising a tetrahydropurine moiety composed of two guanidine units fused together in a stable azaketal linkage, having a molecular formula C10H17N7O2, (mol. wt. 299.30) and to derivatives thereof, including but not limited to hydroxysaxitoxins and neosaxitoxin. Bower et al., Nonprotein Neurotoxins, Clin. Toxicol. 18(7): 813-863 (1981).
Preferred compounds for use in the invention are tetrodotoxin, 4-epi-tetrodotoxin, and anhydro-4-epi-tetrodotoxin.
Routes of administration of tetrodotoxin can include intramuscular injection, intravenous injection, subcutaneous injection, sublingual, patch through the skin, oral ingestion, implantable osmotic pump, collagen implants, aerosols or suppository. The routes of administration, the dosage and the administration schedule are shown in Table 1.
Typically, the active ingredient tetrodotoxin or saxitoxin is formulated into purified water or an acetic acid-sodium acetate buffer as a vehicle. However, the formulation can contain other components, including, but not restricted to, buffering means to maintain or adjust pH, such as acetate buffers, citrate buffers, phosphate buffers and borate buffers; viscosity increasing agents such as polyvinyl alcohol, celluloses, such as hydroxypropyl methyl cellulose and carbomer; preservatives, such as benzalkonium chloride, chlorobutanol, phenylmercuric acetate and phenyl mercuric nitrate; tonicity adjusters, such as sodium chloride, mannitol and glycerine; and penetration enhancers, such as glycols, oleic acid, alkyl amines and the like. The addition of a vasoconstrictor to the formulation is also possible. Combination formulations including the long-acting sodium channel blocking compound and an antibiotic, a steroidal or a non-steroidal anti-inflammatory drug and/or a vasoconstrictor are also possible.
Formulation for each administration route in Table 1 is generally considered known in the art. See, e.g., xe2x80x9cRemington, the Science and Practice of Pharmacyxe2x80x9d, 19th ed., A. R. Gennaro, ed., c. 1995 by The Philadelphia College of Pharmacy and Science, (especially Part 7). As shown in Table 1, the typical dose ranges from 5 to 60 xcexcg per adult. A more typical dose is from 20 to 40 xcexcg per adult.
Tetrodotoxin, its analogs and derivatives are effective in relieving pain in humans and other mammals resulting from malignant neoplasm (cancers) or other tumors. These cancers can occur in the genital organs (including prostate), digestive system (including stomach, colon), breast, respiratory system (including lung and bronchus), urinary system, lymphoma and skin cancer, as shown in the following examples.
Sodium channel blockers are surprisingly shown to be effective as long-term systemic analgesics for alleviation of severe pain. It is also surprising that minimal side effects, the principal one being numbness in the lips and extremities, are observed upon systemic administration. Patients debilitated by pain are able to resume almost normal lives for periods of more than 20 days following a single course of treatment with TTX. That TTX and other sodium channel blockers can be used as systemic analgesics that are more effective than morphine and other opioid analgesics in treating acute, central and chronic pain is entirely unexpected.
An amount of a compound xe2x80x9ceffective for relieving painxe2x80x9d is an amount that results in a decrease in a patient""s perception of pain by 2 units or more on the Numerical Pain Intensity Scale. An amount that is xe2x80x9cvery effective for relieving painxe2x80x9d is an amount that results in a decrease in a patient""s perception of pain by 4 units or more on the Numerical Pain Intensity Scale. An amount of a compound xe2x80x9ceffective for eliminating painxe2x80x9d is an amount that results in a decrease in a patient""s perception of pain to zero on the Numerical Pain Intensity Scale.
1. Ran H P, Bevan S J, Dray A. Nociceptive peripheral neurones: cellular properties. In: Wall P D, Melzack R., editors. Textbook of pain, Edinburgh, chruchill livingstane, 1994; 57-78.
2. Woolf C J, Doubell T P. The pathophysiology of chronic pain-increased sensitivity to low threshold A xcex2-fibre inputs. Current opinion in Neurobiology 1994; 4:525-534.
3. Dray A. Tasting the inflammatory soup: the role of peripheral neurones. Pain Reviews. 1994; 1:153-173.
4. Rabert D K, Koch B D, Ilnicka M, et al. A tetrodotoxin-resistant voltage-gated sodium channel from human dorsal root ganglia, hPH3/SCN 10A. Pain 1998; 78:107-114.
5. Catterall W A, Cellular and molecular biology of voltage-gated sodium channels, Physiol Rev. 1992; 72:s15-s18.
6. Akopian A N, Sivilotti L, Wood J N. A tetrodotoxin-resistant voltage-gated sodium channel expressed by sensory neurons, Nature, 1996; 379:257-262.
7. Gold M S, Reichling D B, Shuster M J, Levine J D. Hyperalgesic agents increase a tetrodotoxin-resistant Na+ current in nociceptors. Prod. Natl Acad Sci. USA, 1996; 93:1108-1112.
8. Khasar S G, Gold M S, Levine J D, A tetrodotoxin-resistant sodium current mediates inflammatory pain in the rat, Neuroscience letters, 1998; 256:17-20.
9. Novakovic S D, Tzoumaka E, McGivern J G, et al. Distribution of the tetrodotoxin-resistant sodium channel PN3 in rat sensory neurons in normal and neuropathic conditions, J. Neuroscience, 1998: 18:2174-2187.
10. Omana-zapata I, Khabbaz M A, Hunter J C, et al. Tetrodotoxin inhibits neuropathil ectopic activity in neuromas, dorsal root ganglia and dorsal horn neurons, Pain, 1997; 72:41-49.